PT - JOURNAL ARTICLE
AU - Ganim, Ziad
AU - Rief, Matthias
TI - Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding
DP - 2017 Oct 03
TA - Proceedings of the National Academy of Sciences
4099 - http://www.pnas.org/content/early/2017/10/02/1704937114.short
4100 - http://www.pnas.org/content/early/2017/10/02/1704937114.full
AB - Green fluorescent protein (GFP) is widely used as a tag to watch otherwise invisible proteins and as a sensor of its local chemical environment. Since GFP can form many partially folded states, it is critical to know how these structural changes affect its signature fluorescence. We use optical tweezers to force single molecules of GFP into folding and unfolding intermediate states and simultaneously probe single-molecule fluorescence from each state. It was found that GFP fluorescence requires complete structural integrity; none of the unfolding or refolding intermediates were observed to fluoresce, but fluorescence could be recovered by complete refolding. This feature was exploited to reversibly, mechanically switch GFP between on and off fluorescence states.Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.